Effect of enzymes on the diauxic lag of denitrifying bacteria switching between electron acceptors

2005·1.3 MB·English

by  Casasus Zambrana, Anna Isabel

EFFECTOFENSZWYIMTECSHIONNGTBHEETDWIEAEUNXIECLELCATGROOFNDAECNCIETPRTIOFRYISNGBACTERIA
By
ANNAISABELCASASUSZAMBRANA
OAFDTIHSESOUEFNRITTVAHETERISROINEDTQPOYURCIOETRFSOEEFRMNLETOONEFRTDIPSDTHAFOIOLITRNOHSPTEOAHGPREHTRDYIAEADGLURFAEUTELEFOSIFCLHLMOEONLT
UNIVERSITYOFFLORIDA
2005
ACKNOWLEDGMENTS
Iwouldlikeloacknowledgemyco-advisors.Dr.SpyrosA,SvoronosandDr.Ben
aKlosoopamcaknn,owfloerdtgheeirthaedovtihseermemnetmabnedtsguoifdmanycecotmhmriotutgeheo.utDrt.hiMsardeeselairncehRparsojcehcct,.DIr.muAsttul
NarangandDr.OscarCrisallc.Itisveryimportanttothankthefellowmembersofmy
trnoeesweeadarrtdochatlthleamanmyk,mgRoyyalafsanmiKin.llyiHffea.omribletionngamnydDinosnpgir-aUtikonLeaendfoprrtohveiidrihneglcpoanntidnsuuepdpomrott.ivFaitniaolnly.I
TABLEOFCONTENTS
AABCSKTNROAWCLTEDGMENTS....
CHAPTER
I IRNETVRIOEDWUOCFTILOITNERATURE
REEPffehffgeeeuncclttoamtooielfonOCnox)onyclgloEemfnngDz.oAyainmurxeNOiislct
OBJECIIVE
ASIMPLEMODEL.
ITFIOTOFENZYMESANDCARBONSIIBSTRATFONTIIFDIAIDCICLAG
CFOWNVOREl.WSOIORNKS. 556?
APPENDIX
AB SSEAMMIP-LLEOGEAXRCIETLHSMPIRCEPALDOSTHSE.ETS - 6M1
CRBIEOFGERRAAEDPNDHCIIETCSIAOLNSAKLEMTOCDHELFITS - - ««»l
EFFECTOFEANboSRsZrWteYtrqIhauMTeciEtCUrnSeHoimfIvOeeDNniNrtGssssiTeBtfrHyoEtrEoaTtftiDhWFoeIlEnoDAEPreUriNgedXrsaIeEeienCLntoEPeLfadCrADttToGioaRclttOOhoFFeNruGloDArffiEaCldPNlCuhImaiEeTtlnPeoRtTsISooOFcfphRYhtoyhSIoeNlGBACTERIA
By
AnnaIsabelCasastisZambrana
May2005
CCDhoeacpiharar:itrm:SepnyBtre:onsCKhAoe.moSipvcmoaarlonnEnogsineering
Thehypothesisthatpresenceofthepcriplasmicnitratereductaseduringaerobic
growthminimizesoreliminatesthediauxiclagwastested.Indeed,littleornolagis
observedwhenbacteriaknowntoexpressthepcriplasmicenzyme(Paracoccus
/tantoirophus)arcsubjectedtoananoxicenvironmentafteraperiodofaerobicgrowth.
Longlagswerestillobservedwhenaerobicallygrownculturesofbacterialackingthe
Therewassomeindicationthatmoreoxidizedsubstratesledtolongerdiauxiclaglengths
forParacoccuspanlolrophus,althoughnolonglagswereobservedforanyofthe
Thepcriplasmicnitratereductaseisnotexpressedinalldenitrilyingbacteria.
theuseofnitrateaselectronacceptor.Asimplemodelhasheenformulatedtosimulate
diauxicgrowthofdenitrifyingbacteriathatonlyhavethemembrane-boundenzyme.It
successfullydepictstheobservedlagswhenapurecultureofPseudomonasdenitrificuns
switchesfromoxygentonitrateasterminalelectronacceptor.Themodelincludesthe
effectofcarbonsubstratelimitationandlengthofaerobicphase;anddocsnotruninto
problemswhenswitchingfromanoxictoaerobicconditions,unlikeearliermodelsof
diauxicgrowth.Inordertolitgrowthdataofbacteriathatalsohavethepcriplosntic
nitratereductase,apreliminarymodelwasdevisedthatincludesthecontributionofboth
INCTHRAOPDTUECRTI1ON
Removalofnitrogencompoundsfromwastewaterisveryimportanttosatisfy
increasinglystringenteffluentqualityrequirements.Therearcseveralreasonswhythisis
anecessaryprocess.Presenceofnitrogencontributestoeutrophicationofbodiesof
water,exertsanoxygendemandthatcancauseharmfuleffectsonaquaticlifeandthe
environment,andistoxicinsomeforms(Ramalho,1983).Biologicalprocessesarethe
preferredmeansofremovingnitrogenfromwastewater(Gradyetal.,1999).
Biologicalnitrogenremovalinwastewatertreatmentplantsisachievedthrough
bacteriallymediatedoxidationofammoniatonitrate;andreductionofnitratetonitrite,
nitrousoxide,nitricoxide,ordinitrogen.Accumulationofanyoftheintermediates(i.c.,
nitrite,nitricoxide,ornitrousoxide)isundesirable.Biologicaloxidationofammoniato
nitrateistermednitrificationandiscarriedoutbyaerobic,autotrophicbacteria(e.g„
NitrosomonasandNitrohacler).Biologicalreductionofnitratetodinilrogenisknownas
completedenitrification.Denitrificationiscarriedoutbyhctcrotrophic,facultative
anaerobicbacteria(e.g..Pseudomonasdenunficans.Paracoccuspamotrophus.
Pseudomonasstutzeri).Althoughdenitrificationunderaerobicconditionshusbeen
nitratepresent)conditions(Ticdjc,1988).Therefore,toachievenitrogenremovalina
single-sludgesystem,amixedpopulationofnitrifyinganddenitrifyingbacteriamustbe
exposedtoalternatingaerobicandanoxicconditions(Ramalho.1983).Inorderforthis
processtobeefficient,thebacteriamustbeabletoadaptquicklytothesechanges.
2002)hSaevveersahloiwnnvetshtaitgwathoerns(baGcotuerwiactcahla.,ng2e00f1r;omLiduiestsoall.v,ed19o9x8yag.e1n99to8bn;itLraitsebaosneclteaclt.r,on
acceptors,theycanexperienceaperiodoflittleornogrowth.Suchaperiod,whenit
occursbetweentwoperiodsofexponentialgrowth,wastermeddiauxiclagbyMonod
(1942).Thesameterminologyisusedinthisstudy.Lagperiods,sincetheyreducethe
overallrateofnitrogenremoval,areundesirableinnitrogen-removingwastewater
treatmentprocesses.Itisbelievedthatduringthislagbacteriawillproducethe
membrane-boundnitratereductase,anenzymethatisinhibitedbyoxygenandis
Someresearchershavereportedsimultaneousutilizationofnitrateandoxygen
(RobertsonandKuencn,1984)byThiosphaerapaniotropha(nowknownasParacoccus
pantolrophus).Thesebacteriaarcknowntohaveapcriplasmicnitratereductase(Nap)
thatisnotinhibitedbyoxygen.Ithasbeensuggestedthatapossibleroleforthisenzyme
istoaidinthetransitionfromenvironmentswhereoxygenispresenttoenvironments
whereitisabsent(Moreno-Vivitaetal.,1999).Ifthisisthecase,thereisapossibility
beensuggestedthatNapisusedforredoxbalancing,andthattheactivityofNap
increaseswiththeextentofreductionofthecarbonsubstrateusedforgrowth
containingbacteriawilldecreaseasthereductionstateofthecarbonsubstrateincreases.
REVIECWHOAFPLTIETRE2RATURE
NitrogenRemoval
Nitrogenremovalcanbeachievedbyseveralphysico-chemicalaswellas
biologicalprocesses.Reactionsgeneratingnitrogen-containinggasescatalyzedby
chemicalagentsareknownaschcmodcnitrificalion.Anexampleoftheseistheacid-
catalyzeddestructionofnitrite,inwhichthepredominantproductisusuallyNO.whichis
undesirable(Ticdje,1988).Thegas.NO.isaveryminorproductofbiological
denitrification,whichisoneofthereasonswhybiologicaldenitrificationisavery
attractivemethodfornitrogenremoval.Furthermore,physico-chemicaltechnologiesare
expensiveanddifficulttomaintain(Gradyetal.,1999).
Biologicalnitrogenremovalfrommunicipalwastewaterrequiresbothnitrification
anddenitrification.Innitrification,ammoniaisoxidizedtonitrite(NOj")inthepresence
do(efNnioOtxfry)ifgiiecnnattbhieoynp,srpneeicstierfnaitcceebioasfcctooexnrvyieagre(tNneidbtyrtooosntoihtmerroonsgapeesnc)igafanisdce.bsai(nctNteOurr.ina,N(jnNiOit.trirtNoe;hi)ascitonextrih)d.eizaIebndsetoncneitorafte
molecularoxygen(Ramalho,1983).
Manyofthebacteriathatcandenitrifyarefacultativeanaerobes,suchas
Pseudomonasdenilriflcans.Thesebacteriacangrowunderbothanoxicandaerobic
conditions(KomarosandLyberatos.1998).However,oxygenrespirationismore
energeticallyefficientthandenitrification(respirationofnitrogenoxides),sofacultative
bacteriausuallyutilizenitrogenoxidesonlyintheabsenceofoxygen(Ohand
Silverstein,1999b).
Anumberofapproachesweretakenwhendevelopingthebasisforbiological
nitrogenremoval.Oneinvolvedtheuseofaseriesofseparatesuspendedgrowthsystems
toaccomplishremovaloforganicmatterandnitrogeninastep-wisefashion.Organic
matterwasremovedinthefirststep,nitrificationoccurredinthesecond,and
dennnficauonoccurredinthethird(withadditionofanexternalcarbonsource).These
systemsinvolvedhighconstructionandoperatingcosts.Anotherapproach,involvingthe
incorporationofaerobiczonesfornitrificationandanoxiczonesfordenitrificationina
singlesystem,withcarbonoxidationoccurringinbothzones,wasdeveloped.Itwas
referredtoassingle-sludgenitrogenremoval(Gradyelal„1999).Inthesesystems,the
bacterialpopulationmustbealternatelyexposedtoaerobicandanoxicenvironments.
Thisforcesthebiomasstoswitchbetweenelectronacceptors,usingoxygenduringthe
aerobicphaseandnitrateintheabsenceofoxygen.
Therequiredsizesoffacilitiesforbiologicaldenitrificationdependonthekinetics
ofdenitrifyingbacteria.Severalfactorsmayaffectdenitrificationkinetics.Forexample,
thequantitiesofnitrogenoxidereductases(enzymesrequiredforutilizationofnitrogen
oxides)areaffectedbyinduction(ifpresent)byNO,'.NO2',NO.andN2O,orrepression
byO2.O2mightdecreaseenzymeactivityduetocompetitionforelectronsorbyenzyme
inactivation.Limitedelectrondonor(shortageofavailablecarbon)mightalsoreduce
enzymeactivity;asmightotherfactors,suchas,pH,temperature,and/ortoxicchemicals
(Tiedje,1988).